Content

Abstract:
Real-time monitoring of biological processes under physiological and pathological conditions is still a great challenge for magnetic resonance imaging (MRI), despite its extensive clinical applications. Calcium(II) is an ideal target for functional MRI purposes, as it is involved in immense number of signaling events in the brain as a secondary messenger. For instance, its extracellular concentration substantially fluctuates during the ischemic stroke [1, 2]. Thus, possibility to track calcium(II) signaling noninvasively would deepen the understanding of numerous physiological processes and allow direct monitoring of neuronal activity.
With that objective, two MR contrast agents bearing same calcium chelating part were employed. They were designed in such a manner that one agent (compound 1) triggers MRI signal changes at variable calcium(II) concentrations, while the other (compound 2) remains calcium-insensitive and does not alter MRI signal at different calcium(II) concentrations. In vitro experiments demonstrated that compound 1 exhibits >90 % increase in longitudinal relaxivity (r1) upon saturation with calcium(II), while r1 of compound 2 remained constant at same conditions, making them ideal candidates for studying the calcium(II) fluctuations as a calcium-responsive and a control MRI agent, respectively.
To monitor calcium(II) changes in vivo, a model of ischemic stroke and the remote middle cerebral artery occlusion (MCAo) approach was used. For these experiments, contrast agents were intracranially injected in Wistar rats (300-340 g), using osmotic pumps for continuous agent delivery (1 μL/h). Thereafter, silicone coated threat (occluder) was introduced through support tubing, and connected with intra-arterial tubing placed inside the common carotid artery. Preparation was completed when occluder was advanced until 2 mm after bifurcation with pterygopalatine artery. First set of MRI acquisitions (7T Bruker BioSpec 70/30 USR) was divided in three parts: pre-ischemia, ischemia, and reperfusion periods, and consisted of acquiring T1-weighted imaging protocol every two minutes. Ischemia was caused and held for 50-60 min by advancing the occluder for 6-8 mm until resistance was felt; later the reperfusion was performed, with reverse occluder actions. Following T1-weighted imaging part, diffusion-weighted and T2-weighted (after more than 4 hours from the onset of ischemia) imaging protocols were acquired to confirm occurrence of stroke. MRI data analysis of acquired T1-weighted signals was based on Kmeans clustering, and obtained results were compared for various numbers of clusters (2-6). The reported MRI signals were normalized to the first acquired signal (at t=0 min).
The in vivo experiments confirmed in vitro responses of employed MR contrast agents. Specifically, T1-weighted images and corresponding masks with 2 clusters show that clusters 1 (cluster with the larger mean value) clearly correspond to center of injections (Figure 1a). Furthermore, the T1-weighted MRI signal of compound 1 varied noticeably because of MCAo stimulation. The MRI signal declined upon MCAo induction, which can be explained through drop of [Ca2+] and accordingly r1 reduction of compound 1. Consequently, r1 recovered upon reperfusion and restoration of [Ca2+], hence also the initial MRI signal trend. On the other hand, the MRI signal of compound 2 changed solely due to continuous injection of the contrast agent, and did not show any alterations to MCAo induction or tissue reperfusion (Figure 1b).
In conclusion, calcium-responsive MRI probes were employed here to demonstrate calcium(II) monitoring in vivo upon the ischemic stroke induction. Considering that neuronal activity is always accompanied with calcium(II) flux, this method yet allows visualization and mapping of neural activity using calcium(II) as its direct indicator. To this end, the introduction of this methodology may circumvent the use of conventional fMRI based on BOLD signal and enable assessment of neuronal activity in direct fashion.